Nucleic acid molecule comprising a nucleic acid sequence coding for a haemocyanin

ABSTRACT

A nucleic acid molecule or construct alone or with a promoter suitable for expression control is contemplated that codes for a HLH2 haemocyanin polypeptide and comprises at least one intron sequence, as well as haemocyanin fusion proteins. The construct further can comprise a nucleic acid sequence that codes for an antigen. Host cells are also contemplated that contain the nucleic acid molecules or construct and recombinant expression product thereof. The invention further relates to a pharmaceutical composition that comprises the expression product of that nucleic acid and antibodies obtainable by immunization of an animal therewith, as well as use of the antibodies in screening methods for the identification of tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 10/049,988 filed Jul. 15,2002 which issued as U.S. Pat. No. 7,125,557 on Oct. 24, 2006, whichapplication claims benefit of PCT application PCT/EP00/08129 filed 21Aug. 2000 which claims benefit of German application No. 199 39 578.0filed 20 Aug. 1999, the contents of which are all incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates to a nucleic acid molecule comprising anucleic acid sequence which codes for a haemocyanin, a haemocyanindomain or a fragment with the immunological properties of at least onedomain of haemocyanin, and comprising at least one intron sequence,constructs which comprise such molecules, host cells which comprise thenucleic acid sequences or the constructs, processes for the preparationof haemocyanin polypeptides, and recombinant haemocyanin polypeptides.

Haemocyanin is a blue copper protein which occurs in a freely dissolvedform in the blood of numerous molluscs and arthropods and transportsoxygen. Of the molluscs, the cephalopods, chitons, most gastropods andsome bivalves contain haemocyanin. Among the arthropods, haemocyanin istypical of arachnids, xiphosurans, malacostracan crustaceans andScutigera. Numerous species of insects contain proteins which arederived from haemocyanin. Haemocyanins are present in the extracellularmedium and float in the haemolymph.

While arthropod haemocyanin has a maximum diameter of 25 nm under anelectron microscope and a subunit has a molecular weight of 75,000 Da,mollusc cyanins are much larger. Thus e.g. the haemocyanin of Megathurahas a diameter of 35 nm and is composed of 2 subunits. Each subunit hasa molecular weight of approx. 400,000 Da and is divided into eightoxygen-binding domains, each of which has a molecular weight of approx.50,000. The domains differ immunologically. These domains can beliberated from the subunit by limited proteolysis.

The haemocyanin of gastropods visible under an electron microscope has amolecular weight of approx. 8 million Da and is a di-decamer. Incontrast to this, the haemocyanin of cephalopods is arranged as anisolated decamer, which also differs significantly from the haemocyaninof gastropods in the quaternary structure.

The haemocyanin of the Californian keyhole limpet Megathura crenulata isof particular immunological interest. The haemocyanin is therefore alsocalled keyhole limpet haemocyanin (KLH). Haemocyanins are very potentantigens. Immunization of a vertebrate leads to a non-specificactivation of the immune system which to date is not very wellunderstood. By the general activation of the immune system, it is thenpossible also to achieve an immune reaction to other foreign structureswhich have previously been tolerated. KLH is used above all as a haptencarrier in order thus to achieve the formation of antibodies against thehapten.

In addition to Megathura crenulata, the abalone Haliotis tuberculataalso belongs to the Archaegastropoda group, which is relatively old inrespect of evolution. It is known that Haliotis also produceshaemocyanin.

KLH is a mixture of two different haemocyanins, which are called KLH1and KLH2. The subunit of KLH1 is a 390 kDa polypeptide which consists ofeight globular domains called 1 a to 1 h according to their sequence inthe subunit. On the other hand, KLH2 has a molecular weight of 350 kDaand according to the most recent data also contains 8 domains, called 2a to 2 h. In vivo every type of subunit forms homo-oligomers, while nohetero-oligomers have been observed.

Amino-terminal, internal and carboxy-terminal domains have been obtainedby limited proteolysis and crossed immunoelectrophoresis of the subunitof KLH1 and KLH2, and their amino-terminal sequences has been determined(Sbhngen et al., Eur. J. Biochem. 248 (1997), 602-614; Gebauer et al.,Zoology 98 (1994), 51-68). However, the resulting sequences do not allowdesigning of sequence-specific primers and/or probes which promisesuccess for hybridization with genomic DNA. Although both KLH types havebeen known since 1991 and 1994 respectively, it has so far not beenpossible to clarify the primary structure.

At the DNA level, in respect of molluscs only the cDNA sequence of thehaemocyanin subunit from the cephalopod Octopus dofleini is so far known(Miller et al., J. Mol. Biol. 278 (1998), 827-842). Octopus dofleini isphylogenetically very far removed from the archaegastropods. Ahaemocyanin gene sequence from molluscs is so far not known at all.

As described by Miller at al. supra, it is difficult both to isolate asingle functional domain (functional unit=domain; also called functionaldomain) and to obtain tissue which is suitable for purification of mRNAfor cDNA sequencing.

There is a further difficulty in the analysis of the haemocyanin fromMegathura crenulata in that the test animals must have reached an age of4 to 8 years for haemolymph to be taken from them in the first place.After the haemolymph has been taken, haemocyanin is not subsequentlyproduced in these animals. It is not yet known how haemocyanin synthesiscould be stimulated. Furthermore, culture of Megathura is extremelyexpensive, since special flow basins are required for this.

BRIEF SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide means andways in order to be able to produce haemocyanin and/or domains thereofin a sufficient amount and inexpensively. This includes the furtherobject of providing a process with which this haemocyanin can beprepared.

This object is achieved according to the invention by a nucleic acidmolecule comprising a nucleic acid sequence which codes for ahaemocyanin, a haemocyanin domain or a functional fragment thereof withthe immunological properties of at least one domain of a haemocyanin,the nucleic acid sequence being selected from

(a) nucleic acid sequences which are selected from the group consistingof the DNA sequences shown below or the corresponding RNA sequences orwhich contain these:

SEQ ID NO:1 (HtH1 domain a+signal peptide),

SEQ ID NO:2 (HtH1 domain b),

SEQ ID NO:3 (HtH1 domain c),

SEQ ID NO:4 (HtH1 domain d),

SEQ ID NO:5 (HtH1 domain e),

SEQ ID NO:6 (HtH1 domain f),

SEQ ID NO:7 (HtH1 domain g),

SEQ ID NO:8 (HtH1 domain h),

SEQ ID NO:9 (partial HtH2 domain b),

SEQ ID NO:10 (HtH2 domain c),

SEQ ID NO:11 (HtH2 domain d),

SEQ ID NO:12 (HtH2 domain e),

SEQ ID NO:13 (HtH2 domain f),

SEQ ID NO:14 (HtH2 domain g),

SEQ ID NO:15 (HtH2 domain h),

SEQ ID NO:16 (partial KLH1 domain b),

SEQ ID NO:17 (KLH1 domain c),

SEQ ID NO:18 (KLH1 domain d),

SEQ ID NO:19 (partial KLH1 domain e),

SEQ ID NO:20 (KLH2 domain b),

SEQ ID NO:21 (KLH2 domain c),

SEQ ID NO:22 (partial KLH2 domain d),

SEQ ID NO:23 (KLH2 domain g),

SEQ ID NO:24 (partial KLH2 domain h),

SEQ ID NO:49 (HtH1 domain a′+signal peptide),

SEQ ID NO:50 (partial HtH2 domain a),

SEQ ID NO:51 (HtH2 domain b′),

SEQ ID NO:52 (HtH2 domain d′),

SEQ ID NO:53 (HtH2 domain e′),

SEQ ID NO:54 (KLH1 domain e′),

SEQ ID NO:55 (KLH1 domain f),

SEQ ID NO:56 (KLH1 domain g),

SEQ ID NO:57 (KLH2 domain b′),

SEQ ID NO:58 (KLH2 domain c′),

SEQ ID NO:59 (KLH2 domain d′),

SEQ ID NO:60 (KLH1 domain e),

SEQ ID NO:61 (KLH2 domain f),

SEQ ID NO:62 (KLH2 domain g′),

SEQ ID NO:80 (HtH1 domain a″+signal peptide),

SEQ ID NO:81 (HtH1 domain b″),

SEQ ID NO:82 (HtH1 domain c″),

SEQ ID NO:83 (HtH1 domain d″),

SEQ ID NO:84 (HtH1 domain e″),

SEQ ID NO:85 (HtH1 domain f″),

SEQ ID NO:86 (HtH1 domain g″),

SEQ ID NO:87 (HtH1 domain h″),

SEQ ID NO:88 (partial HtH2 domain a″),

SEQ ID NO:89 (HtH2 domain b″),

SEQ ID NO:90 (HtH2 domain c″),

SEQ ID NO:91 (HtH2 domain d″),

SEQ ID NO:92 (HtH2 domain e″),

SEQ ID NO:93 (HtH2 domain f″),

SEQ ID NO:94 (HtH2 domain g″),

SEQ ID NO:95 (HtH2 domain h″),

SEQ ID NO:96 (partial KLH1 domain b″),

SEQ ID NO:97 (KLH1 domain c″),

SEQ ID NO:98 (KLH1 domain d″),

SEQ ID NO:99 (KLH1 domain e″),

SEQ ID NO:100 (KLH1 domain f″),

SEQ ID NO:101 (KLH1 domain g″),

SEQ ID NO:102 (KLH2 domain b″),

SEQ ID NO:103 (KLH2 domain c″),

SEQ ID NO:104 (KLH2 domain d″),

SEQ ID NO:105 (KLH2 domain e″),

SEQ ID NO:106 (KLH2 domain f″),

SEQ ID NO:107 (KLH2 domain g″),

SEQ ID NO:108 (partial KLH2 domain h″),

SEQ ID NO:157 (complete HtH2 domain a),

b) nucleic acid sequences which hybridize with the counter-strand of anucleic acid sequence according to (a) and code for a polypeptide whichhas the immunological properties of at least one domain of ahaemocyanin;

(c) nucleic acid sequences which on the basis of the genetic code aredegenerated to the DNA sequences defined under (a) and (b) and code fora polypeptide which has the immunological properties of at least onedomain of a haemocyanin;

(d) nucleic acid sequences which hybridize with one of the nucleic acidsequences described under (a) to (c) and the counter-strand of whichcodes for a polypeptide which has the immunological properties of atleast one domain of a haemocyanin;

(e) nucleic acid sequences which are at least 60% homologous to one ofthe nucleic acid sequences described under (a);

(f) variants of the sequences described under (a) to (e), the variantscontaining additions, deletions, insertions or inversions and coding fora polypeptide which has the immunological properties of at least onedomain of haemocyanin; and

(g) combinations of several of the DNA sequences described under (a) to(f).

Preferably, the intron sequence is selected from the followingsequences:

(i) nucleic acid sequences which are selected from the group consistingof the DNA sequences shown below or the corresponding RNA sequences orwhich contain these:

SEQ ID NO:109 (HtH1 intron 1S-1/1S-2),

SEQ ID NO:110 (HtH1 intron 1S-2/1A-1),

SEQ ID NO:111 (HtH1 intron 1A-1/1A-2),

SEQ ID NO:112 (HtH1 intron 1A-2/1A-3),

SEQ ID NO:113 (HtH1 intron 1A-3/1A-4),

SEQ ID NO:114 (HtH1 intron 1A-4/1B),

SEQ ID NO:115 (HtH1 intron 1B/1C),

SEQ ID NO:116 (HtH1 intron 1C/1D),

SEQ ID NO:117 (HtH1 intron 1D/1E),

SEQ ID NO:118 (HtH1 intron 1E/1F-1),

SEQ ID NO:119 (HtH1 intron 1F-1/1F-2),

SEQ ID NO:120 (HtH1 intron 1F-2/1G-1),

SEQ ID NO:121 (HtH1 intron 1F-1/1G-2),

SEQ ID NO:122 (HtH1 intron 1G-2/1G-3),

SEQ ID NO:123 (HtH1 intron 1G-3/1H),

SEQ ID NO:124 (HtH1 intron in the 3′UTR of HtH1;

SEQ ID NO:125 (HtH2 intron 2A-1/2A-2);

SEQ ID NO:126 (HtH2 intron 2A-1/2A-3),

SEQ ID NO:127 (HtH1 intron 2A-1/2A-4),

SEQ ID NO:128 (HtH1 intron 2A-4/2B),

SEQ ID NO:129 (HtH1 intron 2B/2C),

SEQ ID NO:130 (HtH1 intron 2C/2D),

SEQ ID NO:131 (HtH1 intron 2D/2E),

SEQ ID NO:132 (HtH1 intron 2F-1/2F-2),

SEQ ID NO:133 (HtH1 intron 2F-2/2GF-1),

SEQ ID NO:134 (HtH1 intron 2F-2/2GF-1),

SEQ ID NO:135 (HtH1 intron 2G-1/2G-2),

SEQ ID NO:136 (HtH1 intron 2G-2/2G-3),

SEQ ID NO:137 (HtH1 intron 2G-3/2H),

SEQ ID NO:138 (HtH1 intron in the 3′UTR of HtH2),

SEQ ID NO:139 (KLH1 intron 1B/1C),

SEQ ID NO:140 (KLH1 intron 1C/1D),

SEQ ID NO:141 (KLH1 intron 1D/1E),

SEQ ID NO:142 (KLH1 intron 1E/1F),

SEQ ID NO:143 (KLH1 intron 1F-1/2F-2),

SEQ ID NO:144 (KLH1 intron 1F-2/1G-1),

SEQ ID NO:145 (KLH1 intron 1G-1/1G-2),

SEQ ID NO:146 (KLH1 intron 1G-2/1G-3),

SEQ ID NO:147 (KLH1 intron 2B/2C),

SEQ ID NO:148 (KLH2 intron 2C/2D),

SEQ ID NO:149 (KLH2 intron 2D/2E),

SEQ ID NO:150 (KLH2 intron 2E/2F),

SEQ ID NO:151 (KLH2 intron 2F),

SEQ ID NO:152 (KLH2 intron 2F-2/2G),

SEQ ID NO:153 (KLH2 intron 2G-2/2G-2),

SEQ ID NO:154 (KLH2 intron 2G-2/2G-3),

SEQ ID NO:155 (KLH2 intron 2G/2H);

(ii) nucleic acid sequences which hybridize with the counter-strand of anucleic acid sequence according to (1);

(iii) nucleic acid sequences which are at least 60% homologous to one ofthe nucleic acid sequences described under (i);

(vi) variants of the sequences described under (i) to (iii), thevariants containing additions, deletions, insertions or inversions withrespect to the sequences described under (i) to (iii); and

(v) combinations of several of the DNA sequences described under (i) to(iv).

Some terms are explained in more detail below in order to clarify howthey are to be understood in connection with the present application.

The term “haemocyanin” as used below in the description includescomplete haemocyanin, haemocyanin domains and/or fragments, haemocyaninmutants and fusion proteins. In respect of fusion proteins, theseinclude, in particular, those in which the fusion comprises haemocyaninand antigens.

“Domains” are understood as meaning functional partial sequences of thehaemocyanin subunits which can be separated from one another, forexample, by limited proteolysis. They can furthermore have differentimmunological properties.

The “immunological properties of at least one domain of haemocyanin”means the property of a polypeptide of inducing, in the same manner asat least one domain of haemocyanin, an immunological response of therecipient immunized with the polypeptide. “Immunological response” hereis understood as meaning T and/or B cell responses to haemocyaninepitopes, such as, for example, an antibody production. Theimmunological reaction can be observed, for example, by immunization ofa mammal, such as e.g. a mouse, a rat or a rabbit, with thecorresponding polypeptide and comparison of the immune response to thepolypeptide used for the immunization with the immune response tonatural haemocyanins.

The term “intron sequence” refers either to a sequence interrupting aneukaryotic gene or to the corresponding sequence in the RNA transcript.The intron sequence(s) and the coding sequence(s) are transcribedtogether; the intron transcript or transcripts are then deleted toobtain the functional RNA.

According to the invention, the term “antigen” includes both haptens andweak and potent antigens. Haptens are characterized in that they aresubstances of low molecular weight (less than 4,000 Da), but withoutbeing coupled to a carrier molecule are not capable of inducing animmunological reaction. Weak antigens are substances which canthemselves already induce an immunological reaction and of which thepotential to be able to induce an immunological reaction can beincreased further by coupling with a carrier molecule at the proteinand/or DNA level.

“H is tag” means a sequence of at least 6 histidine amino acids which,by corresponding cloning and fusion with an expressible sequence, leadsto a fusion protein which has at least 6 H is residues on the NH₂terminus and can easily be purified by complexing with an Ni²⁺ column.

“Cloning” is intended to include all cloning methods known in the priorart which could be employed here but which are not all described indetail because they belong to the obvious hand tools of the skilledperson.

“Variants” of a nucleic acid sequences include additions, deletions,insertions or inversions and code for a polypeptide which has theimmunological properties of at least one domain of a haemocyanin.Variants can be synthetic or natural. Allelic variants are an example ofnatural variants.

“Recombinant expression in a suitable host cell” is to be understood asmeaning all the expression methods known in the prior art in knownexpression systems which could be employed here but which are not alldescribed in detail because they belong to the obvious hand tools of theskilled person.

The nucleic acid sequence contained in the nucleic acid moleculeaccording to the invention can be genomic DNA, cDNA or synthetic DNA,synthetic DNA sequences also being understood as meaning those whichcomprise modified internucleoside bonds. The nucleic acid sequences canfurthermore be RNA sequences, which may be necessary e.g. for expressionby means of recombinant vector systems. The nucleic acid sequencesaccording to (b) are obtainable, for example, by using a detectablymarked probe which corresponds to one of the sequences described under(a) or a fragment, or a counter-strand thereof for screeningcDNA/genomic DNA libraries from molluscs or arthropods. The mRNA onwhich the cDNA library is based is preferably to be obtained frommollusc tissues which express haemocyanin to a particularly high degree,such as e.g. mantle tissue from gastropods and branchial gland tissuefrom cephalopods.

Positive cDNA/genomic DNA clones are identified by standard methods. Cf.Maniatis et al., Molecular Cloning (1989) Cold Spring Harbor LaboratoryPress.

In a preferred embodiment, the hybridization described under (b) or (d)is carried out under stringent conditions. Stringent hybridizationconditions are e.g. 68° C. overnight in 0.5×SSC; 1% blocking reagent(Boehringer Mannheim); 0.1% sodium lauryl sarcosinate and subsequentwashing with 2×SSC; 0.1% SDS.

In a preferred embodiment, nucleic acid sequences which are at least 60%homologous to one of the nucleic acid sequences described under (a) areprovided. The nucleic acid sequences are preferably at least 80%homologous to one of the nucleic acid sequences described under (a). Thenucleic acid sequences are particularly preferably at least 90%homologous to one of the nucleic acid sequences described under (a). Inparticular, the nucleic acid sequences are at least 95% homologous toone of the nucleic acid sequences described under (a).

In a further preferred embodiment, nucleic acid sequences comprising atleast one intron sequence which is at least 60% homologous to one of thenucleic acid sequences described under (i) are provided. Intronsequence(s) which are at least 80% homologous to one of the nucleic acidsequences described under (i) are preferred. Intron sequence(s) whichare at least 90% homologous to one of the nucleic acid sequencesdescribed under (i) are particularly preferred. In particular, theintron sequence(s) are at least 95% to one of the nucleic acid sequencesdescribed under (i).

According to the invention, the term “homology” means homology at theDNA level, which can be determined by known methods, e.g.computer-assisted sequence comparisons (Basic local alignment searchtool, S. F. Altschul et al., J. Mol. Biol. 215 (1990), 403-410).

The term “homology” known to the skilled person describes the degree towhich two or more nucleic acid molecules are related, this beingdetermined by the concordance between the sequences. The percentage of“homology” is obtained from the percentage of identical regions in twoor more sequences, taking into account gaps or other sequencepeculiarities.

The homology of nucleic acid molecules which are related to one anothercan be determined with the aid of known methods. As a rule, specialcomputer programs with algorithms which take account of the particularrequirements are employed.

Preferred methods for the determination of homology initially producethe greatest concordance between the sequences analysed. Computerprograms for determination of the homology between two sequencesinclude, but are not limited to, the GCG program package, including GAP(Devereux, J., et al., Nucleic Acids Research 12 (12): 387 (1984);Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP,BLASTN and FASTA (Altschul, S. et al., J. Mol. Biol. 215:403-410(1990)). The BLASTX program can be obtained from the National Centre forBiotechnology Information (NCBI) and from other sources (BLAST Handbook,Altschul S., et al., NCB NLM NIH Bethesda Md. 20894; Altschul, S., etal., J. Mol. Biol. 215:403-410 (1990)). The known Smith Watermanalgorithm can also be used for determining homologies.

Preferred parameters for the comparison of nucleic acid sequencesinclude the following:

Algorithm: Needeman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: Concordance (matches) = +10 Non-concordance(mismatch) = 0 Gap penalty: 50 Gap length penalty: 3

The GAP program is also suitable for use with the above parameters. Theabove parameters are the default parameters for nucleic acid sequencecomparisons.

Further algorithms, gap opening penalties, gap extension penalties andcomparison matrices by way of example, including those mentioned in theProgram Handbook, Wisconsin Package, version 9, September 1997, can beused. The choice depends on the comparison to be made and furthermore onwhether the comparison is to be made between sequence pairs, in whichcase GAP or Best Fit are preferred, or between a sequence and acomprehensive sequence databank, in which case FASTA or BLAST arepreferred.

A concordance of 60% determined with the abovementioned algorithm isdesignated 60% homology in the context of this application. The sameapplies accordingly to higher degrees of homology.

In a preferred embodiment, the DNA sequence according to the inventionis a combination of several of the DNA sequences described under (a) to(f), which can be obtain by fusion and optionally cloning, which areknown to the skilled person. These combinations are of particularinterest, since they are particularly immunogenic. Combinations whichcontain several or all of the domains in the sequence (a to h) whichoccurs naturally in the subunit are particularly preferred. Embodimentsin which the nucleic acid sequences which code for the domains arecoupled to one another directly in frame are particularly preferred.

Constructs which comprise the nucleic acid molecules according to theinvention are furthermore provided. In a preferred embodiment, theconstruct according to the invention comprises a promoter which issuitable for expression, the nucleic acid sequence being under thecontrol of the promoter. The choice of promoter depends on theexpression system used for expression. Generally, constitutive promotersare preferred, but inducible promoters, such as e.g. the metallothioneinpromoter, are also possible.

In a further preferred embodiment, the construct furthermore comprisesan antigen-coding nucleic acid sequence which is bonded directly to thehaemocyanin nucleic acid according to the invention. The antigen-codingsequence can be located both 5′ and 3′ relative to the haemocyaninsequence or also on both ends. It either follows the haemocyaninsequence directly in the same reading frame, or is coupled to it by anucleic acid linker, the reading frame being preserved. By fusion of theantigen-coding sequence with the haemocyanin sequence the formation of afusion protein in which the antigen-coding sequence is bonded covalentlyto the haemocyanin sequence is intended. The antigen according to theinvention is a medically relevant antigen, which is selected, forexample, from: tumour antigens, virus antigens and antigens of bacterialor parasitic pathogens. Tumour antigens can be, for example, Rb and p53.The virus antigens preferably originate from immunologically relevantviruses, such as e.g. influenza virus, hepatitis virus and HIV. Pathogenantigens are, inter alia, those from mammalian pathogens, in particularorganisms which are pathogenic to humans, such as e.g. Plasmodium.Bacterial antigens can originate e.g. from Klebsiella, Pseudomonas, E.coli, Vibrio cholerae, Chlamydia, Streptococci or Staphylococci.

In another preferred embodiment, the construct furthermore comprises atleast a part of a vector, in particular regulatory regions, the vectorbeing selected from: bacteriophages, such as □ derivatives,adenoviruses, vaccinia viruses, baculoviruses, SV40 viruses andretroviruses, preferably MoMuLV (Moloney murine leukaemia virus).

A construct which additionally comprises a H is tag-coding DNA sequence,which, when expressed, leads to the formation of a fusion protein with aH is tag on the NH₂ terminus of the haemocyanin, facilitatingpurification of the protein on a nickel column by chelate formation, isfurthermore preferred.

The invention furthermore provides host cells which contain theconstruct and which are suitable for expression of the construct.Numerous prokaryotic and eukaryotic expression systems are known in theprior art, the host cells being selected, for example, from prokaryoticcells, such as E. coli or B. subtilis, from eukaryotic cells, such asyeast cells, plant cells, insect cells and mammalian cells, e.g. CHOcells, COS cells or HeLa cells, and derivatives thereof. For examplecertain CHO production lines of which the glycosylation patterns arealtered compared with CHO cells are known in the prior art. Thehaemocyanins obtained using glycosylation-deficient orglycosylation-reduced host cells possibly have additional epitopes whichare otherwise not accessible to the immune system of the recipient inthe case of complete glycosylation, so that haemocyanins with a reducedglycosylation under certain circumstances have an increasedimmunogenicity. From plant cells transformed with the constructaccording to the invention it is possible to produce transgenic plantsor plant cell cultures which produce haemocyanin polypeptides, forexample tobacco, potato, tomato, sugar beet, soya bean, coffee, pea,bean, rape, cotton, rice or maize plants or plant cell cultures.

The present invention also relates to a process for the preparation of ahaemocyanin polypeptide. For this, the nucleic acid molecule accordingto the invention and/or the construct is expressed in a suitable hostcell and the protein is isolated from the host cell or the medium bymeans of conventional processes.

Numerous processes for expression of DNA sequences are known to theskilled person; compare Recombinant Gene Expression Protocols in Methodsin Molecular Biology, volume 62, Humana Press Totowa N.J. (1995). Theexpression can be both constitutive and inducible, inducers such as, forexample, IPTG and Zn²⁺ being known to the skilled person. If a H is taghas been fused on to the NH₂ terminus of the haemocyanin, thehaemocyanin prepared can be purified by chelate formation on a nickelcolumn. Processes for the purification of haemocyanin, in particularKLH, are to be found in Harris et al., Micron 26 (1995), 201-212. Thehaemocyanin is preferably purified by ion exchange chromatography and/orgel filtration chromatography. The procedure for these measures is knownto the skilled person.

In another preferred embodiment, the haemocyanin prepared according tothe invention is modified. The modifications include di-, oligo- andpolymerization of the monomeric starting substance, for example bycrosslinking, e.g. by means of dicyclohexylcarbodiimide or pegylation orassociation (self assembly). The di-, oligo- and polymers prepared inthis way can be separated from one another by gel filtration. Theformation of decamers, didecamers or multidecamers is intended inparticular. Further modifications include side chain modifications, forexample of ε-amino-lysine residues of the haemocyanin, or amino- orcarboxy-terminal modifications. Modification of the haemocyanin bycovalent bonding to an antigen is particularly preferred, it beingpossible for the antigen to be reacted stoichiometrically ornon-stoichiometrically with the haemocyanin. The antigen is preferablyselected from tumour antigens, virus antigens and pathogen antigens, asmentioned above. Further modifications include post-translationalevents, e.g. glycosylation or partial or complete deglycosylation of theprotein.

In a preferred embodiment, the haemocyanin obtained by recombinantexpression in prokaryotes or glycosylation-deficient eukaryotes isnon-glycosylated. Haemocyanin which is glycosylated by recombinantexpression in eukaryotes which are capable of glycosylation, such asyeast cells, plant cells, insect cells or mammalian cells, such as CHOcells or HeLa cells, is also possible according to the invention.

Haemocyanin polypeptides which comprise an amino acid sequence, theamino acid sequence being coded by one or more of the nucleic acidmolecules according to the invention, are provided in anotherembodiment,

Haemocyanin polypeptides which comprise at least one amino acid sequenceselected from the following group:

SEQ ID NO:25 (HtH1 domain a+signal peptide),

SEQ ID NO:26 (HtH1 domain b),

SEQ ID NO:27 (HtH1 domain c),

SEQ ID NO:28 (HtH1 domain d),

SEQ ID NO:29 (HtH1 domain e),

SEQ ID NO:30 (HtH1 domain f),

SEQ ID NO:31 (HtH1 domain g),

SEQ ID NO:32 (HtH1 domain h),

SEQ ID NO:33 (partial HtH2 domain b),

SEQ ID NO:34 (HtH2 domain c),

SEQ ID NO:35 (HtH2 domain d),

SEQ ID NO:36 (HtH2 domain e),

SEQ ID NO:37 (HtH2 domain f),

SEQ ID NO:38 (HtH2 domain g),

SEQ ID NO:39 (HtH2 domain h),

SEQ ID NO:40 (partial KLH1 domain b),

SEQ ID NO:41 (KLH1 domain c),

SEQ ID NO:42 (partial KLH1 domain d),

SEQ ID NO:43 (partial KLH1 domain e),

SEQ ID NO:44 (KLH2 domain b),

SEQ ID NO:45 (KLH2 domain c),

SEQ ID NO:46 (partial KLH2 domain d),

SEQ ID NO:47 (KLH2 domain g),

SEQ ID NO:48 (partial KLH2 domain h),

SEQ ID NO:63 (HtH1 domain a′+signal peptide),

SEQ ID NO:64 (HtH1 domain h′),

SEQ ID NO:65 (partial HtH2 domain a),

SEQ ID NO:66 (HtH2 domain b′),

SEQ ID NO:67 (HtH2 domain d′),

SEQ ID NO:68 (HtH2 domain e′),

SEQ ID NO:69 (partial KLH1 domain b′),

SEQ ID NO:70 (KLH1 domain e′),

SEQ ID NO:71 (KLH1 domain f),

SEQ ID NO:72 (KLH1 domain g),

SEQ ID NO:73 (KLH1 domain h),

SEQ ID NO:74 (KLH2 domain b′),

SEQ ID NO:75 (KLH2 domain c′),

SEQ ID NO:76 (KLH2 domain d′),

SEQ ID NO:77 (KLH2 domain e),

SEQ ID NO:78 (KLH2 domain f),

SEQ ID NO:79 (KLH2 domain g′),

SEQ ID NO.:158 (partial KLH2 domain h),

or a fragment of one of these sequences which has the immunologicalproperties of at least one domain of haemocyanin are preferred.

The invention also includes haemocyanin polypeptides of which thesequence shows at least 60% or 70%, preferably at least 80%,particularly preferably at least 90% or 95% homology to one of the aminoacid sequences according to SEQ ID NO:25 to 48 and SEQ ID NO:63 to 79over a partial region of at least 90 amino acids.

In this connection, the expression “at least 70%, preferably at least80%, particularly preferably at least 90% homology” relates toconcordance at the amino acid sequence level, which can be determined byknown methods, e.g. computer-assisted sequence comparisons (Basic localalignment search tool, S. F. Altschul et al., J. Mol. Biol. 215 (1990),403-410).

The term “homology” known to the skilled person describes here thedegree to which two or more polypeptide molecules are related, thisbeing determined by the concordance between the sequences, concordancebeing understood as meaning both identical concordance and conservativeamino acid exchange. The percentage of “homology” is obtained from thepercentage of regions in concordance in two or more sequences, takinginto account gaps or other sequence peculiarities.

The expression “conservative amino acid exchange” relates to an exchangeof an amino acid residue for another amino acid residue, where theexchange does not lead to a change in polarity or charge. An example ofa conservative amino acid exchange is the exchange of a non-polar aminoacid residue for another non-polar amino acid residue.

The homology of polypeptide molecules which are related to one anothercan be determined with the aid of known methods. As a rule, specialcomputer programs with algorithms which take account of the particularrequirements are employed. Preferred methods for the determination ofhomology initially produce the greatest concordance between thesequences analysed. Computer programs for determination of the homologybetween two sequences include, but are not limited to, the GCG programpackage, including GAP (Devereux, J., et al., Nucleic Acids Research 12(12): 387 (1984); Genetics Computer Group University of Wisconsin,Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul, S. et al., J. Molec.Biol. 215:403/410 (1990)). The BLAST X program can be obtained from theNational Centre for Biotechnology Information (NCBI) and from othersources (BLAST Handbook, Altschul S., et al., NCB NLM NIH Bethesda Md.20894; Altschul, S., et al., J. Mol. 215:403/410 (1990)). The knownSmith Waterman algorithm can also be used for determining homology.

Preferred parameters for the sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol 48:443-453 (1970)Comparison matrix: BLOSUM 62 of Henikoff and Henikoff, Proc. Natl. Acad.Sci. USA 89:10915-10919 (1992) Gap penalty: 12 Gap length penalty: 4Similarity threshold: 0

The GAP program is also suitable for use with the above parameters. Theabove parameters are the standard parameters (default parameters) foramino acid sequence comparisons where gaps at the ends do not reduce thehomology value. If sequences are very short compared with the referencesequence, it may furthermore be necessary to increase the expected valueto up to 100,000 and where appropriate to reduce the word size down to2.

Further algorithms, gap opening penalties, gap extension penalties andcomparison matrices by way of example, including those mentioned in theProgramm-Handbuch, Wisconsin-Paket [Program Handbook, WisconsinPackage], version 9, September 1997, can be used. The choice depends onthe comparison to be made and furthermore on whether the comparison isto be made between sequence pairs, in which case GAP or best fit arepreferred, or between a sequence and a comprehensive sequence database,in which case FASTA or BLAST are preferred.

A concordance of 60% determined with the above mentioned algorithm isdesignated 60% homology in the context of this Application. The sameapplies accordingly to higher degrees of homology.

In another embodiment, the invention provides haemocyanin polypeptideswhich are obtainable by the recombinant preparation method ormodifications thereof.

Preferred haemocyanin polypeptides are those which comprise each of thesequences SEQ ID NO: 25 to 32, it being possible for the sequence withSEQ ID NO:25 to be replaced by SEQ ID NO:63 and/or SEQ ID NO:32 to bereplaced by SEQ ID NO:64. Haemocyanin polypeptides which are alsopreferred are those which comprise either the sequences SEQ. ID NO: 33to 39 or the sequences SEQ ID NO:65, 66, 34-39, it being possible forSEQ ID NO:35 to be replaced by SEQ ID NO:67 and/or SEQ ID NO:36 to bereplaced by SEQ ID NO:68. These haemocyanin polypeptides areparticularly preferably haemocyanin 1 or 2 from Haliotis tuberculata.

Haemocyanin 1 from Haliotis tuberculata, which has an apparent molecularweight of 370 kDa in SDS-PAGE under reducing conditions, is particularlypreferred. Haemocyanin 2 from Haliotis tuberculata, which has anapparent molecular weight of 370 kDa in SDS-PAGE under reducingconditions, is furthermore particularly preferred. The haemocyanins areobtainable from whole haemocyanin from Haliotis tuberculata by theselective dissociation process described in the examples.

Haemocyanin polypeptides which are furthermore preferred are those whichcomprise each of the sequences SEQ ID NO: 40 to 43 or the sequences SEQID NO:40 to 43 and SEQ ID NO:71 to 73, it being possible in each casefor the sequence with SEQ ID NO:40 to be replaced by SEQ ID NO:66 and/orSEQ ID NO:43 to be replaced by SEQ ID NO:70. Haemocyanin polypeptideswhich are also preferred are those which comprise either each of thesequences SEQ ID NO: 44 to 48 or the sequences SEQ ID NO:44 to 46, 77,78, 47, 48, it being possible in each case for the sequence with SEQ IDNO:44 to be replaced by SEQ ID NO:74, SEQ ID NO:45 to be replaced by SEQID NO:75, SEQ ID NO:46 to be replaced by SEQ ID NO:76 and/or SEQ IDNO:47 to be replaced by SEQ ID NO:79.

These haemocyanin polypeptides are particularly preferably completehaemocyanin 1 (KLH1) or 2 (KLH2) from Megathura crenulata.

Non-glycosylated and glycosylated haemocyanin polypeptide obtainable byexpression in host cells which are capable or incapable of glycosylationis furthermore provided. Depending on the envisaged use of thehaemocyanin polypeptide, the glycosylation pattern of yeast, inparticular methylotrophic yeast, of plant cells or of COS or HeLa cellscan be preferred.

The invention furthermore relates to pharmaceutical compositions whichcomprise the nucleic acid molecules according to the invention andphysiologically tolerated additives known in the prior art. Thepharmaceutical compositions are preferably employed for non-specificimmunostimulation in the form of a gene therapy, haemocyaninpolypeptides being expressed after transformation with a suitable vectorand serving to antigenize the tissue.

In particular, the invention provides the use of a nucleic acid moleculeaccording to the invention which is bonded to an antigen-coding DNAsequence for specific immunization against this antigen. Without beingbound to this theory, the immunization here is based on non-specificstimulation of the immune system by haemocyanin polypeptide epitopes andmore extensive specific immunization by recognition of antigen epitopesby the immune system.

Such an immunization is particularly valuable in respect of pathogenantigens, and especially in respect of tumour antigens. The usability ofthe pharmaceutical composition according to the invention for treatmentof tumour diseases also results from the cross-reactivity of thehaemocyanin-specific antibodies with carbohydrate residues, which occuron the surface of tumours, such as e.g. the Thomsen-Friedenreichantigen, which occurs in the majority of human tumours, such asepithelial carcinomas, ovarian carcinoma, colorectal carcinoma, mammarycarcinoma, bronchial carcinoma and bladder carcinoma.

The pharmaceutical compositions according to the invention canfurthermore be employed for treatment of parasitic diseases, such asschistosomiasis, and for prevention of cocaine abuse.

Pharmaceutical compositions which comprise a haemocyanin polypeptideaccording to the invention in combination with one or morephysiologically tolerated additives are provided as a further embodimentof the present invention. As already mentioned above, such a haemocyaninpolypeptide can consist of a complete haemocyanin subunit, of one ormore domains and of one or more fragments of such domains, provided thatthese fragments still have the immunological properties of at least onedomain of a haemocyanin. Such a pharmaceutical composition is suitablee.g. as an antiparasitic composition, antivirus composition orantitumour composition due to either the non-specific immunostimulation,which is to be attributed solely to the haemocyanin, or due to thespecific immune reaction to antigens associated with the haemocyanin. Itcan thus be employed e.g. for treatment of schistosomiasis, epithelialcarcinomas, ovarian carcinoma, colorectal carcinoma, mammary carcinoma,bronchial carcinoma and bladder carcinomas, but is also suitable fortreatment of high blood pressure. The treatment of high blood pressureis achieved by carrying out an immunization with the aid ofhaemocyanin-β-adrenergic receptor peptide constructs and/or fusionproteins.

In another embodiment, the pharmaceutical compositions according to theinvention are used as vaccines. They can thus make a valuablecontribution to the prophylaxis of diseases caused by known pathogens.This applies in particular to pharmaceutical compositions in which ahaemocyanin polypeptide is coupled to a virus, virus constituent, killedbacteria, bacteria constituents, in particular surface proteins fromvirus or bacteria envelopes, DNA, DNA constituents, inorganic or organicmolecules, e.g. carbohydrates, peptides and/or glycoproteins.

According to another preferred embodiment, the pharmaceuticalcomposition according to the invention is used for prevention of cocaineabuse.

Liposomes are particularly suitable for administration both of thenucleic acid molecules according to the invention and of the haemocyaninpolypeptides. The present invention accordingly relates to liposomeswhich comprise a nucleic acid molecule according to the invention, aconstruct according to the invention or a haemocyanin polypeptideaccording to the invention.

Various methods for the preparation of liposomes which can be used forpharmaceutical purposes are known to the skilled person. The selectivityof the liposomes comprising the nucleic acid molecules or haemocyaninpolypeptides according to the invention can be increased by theadditional incorporation into the liposome of cell recognitionmolecules, which bind selectively to target cells. Receptor ligandswhich bind to receptors of the target cells or, especially in the caseof tumours, antibodies directed against surface antigens of theparticular target cells envisaged are particularly suitable for this.

The haemocyanin polypeptides according to the invention are furthermoreenvisaged as carrier molecules for medicaments, such as e.g.cytostatics. The increase in the molecular weight prolongs thephysiological half-life of the medicaments considerably since the lossdue to ultrafiltration in the kidneys is significantly reduced.

The vaccines are formulated by methods known to the skilled person; insome embodiments the additional use of adjuvants, such as e.g. Freund'sadjuvant or polysaccharides, is envisaged.

The invention furthermore provides antibodies which react specificallywith the haemocyanin polypeptide according to the invention and areobtainable by immunization of a test animal with a haemocyaninpolypeptide. Polyclonal antibodies can be obtained by immunization, forexample, of rabbits and subsequent isolation of antisera. Monoclonalantibodies can be obtained by standard methods by immunization of e.g.mice, isolation and immortalization of the spleen cells and cloning ofthe hybridomas which produce antibodies specific for haemocyanin.

A screening method for identification of tumour-specific DNA in a cellis furthermore provided, this comprising the steps:

a) bringing cell DNA and/or cell protein into contact with a probecomprising the nucleic acid molecule according to the invention and/orthe antibody according to the invention and

b) detecting the specific binding.

The tumour to be detected is preferably a bladder carcinoma, epithelialcarcinoma, ovarian carcinoma, mammary carcinoma, bronchial carcinoma orcolorectal carcinoma.

It is intended to illustrate the invention with the following figuresand examples, but not to limit this in any way. Further embodiments,which are also included, are accessible to the skilled person on thebasis of the description and the examples.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the characterization and purification of Haliotistuberculata haemocyanin (HtH):

(a) Electron microscopy of negatively stained whole HtH, which has beenpurified by ultracentrifugation of cell-free haemolymph;

(b) SDS polyacrylamide gel electrophoresis (7.5% polyacrylamide) of HtH1compared with KLH (MW 370 kDa);

(c) Native polyacrylamide gel electrophoresis (5% polyacrylamide) of theHtH subunit preparation, the anode being at the lower edge;

(d) Crossed immunoelectrophoresis of the two HtH subunits using anti-HtHantibodies from the rabbit;

(e) Electron microscopy of the remaining HtH1 didecamers (white arrows)after selective dissociation of HtH2 (black arrows);

(f) Elution profile of the gel filtration chromatography (Biogel A15m)in the presence of ammonium molybdate/polyethylene glycol solution (pH5.9) after selective dissociation of HtH2 into its subunit andsubsequent concentration of HtH1 by ultracentrifugation;

(g) Native polyacrylamide gel electrophoresis (6.5% polyacrylamide) ofHtH1 and HtH2 subunits purified by gel chromatography compared with thestarting material;

(h,i) Crossed immunoelectrophoresis of chromatographically purified HtHsubunits; and

(j,m) Crossed immunoelectrophoresis of the purified HtH subunits usinganti-KLH antibodies from the rabbit which are specific for KLH1 andKLH2.

FIG. 2 shows the analysis of the subunit organization of HtH1, anti-HtH1antibodies from the rabbit having been used for theimmunoelectrophoresis and the anode being on the left-hand side;

(a) Crossed immunoelectrophoresis after limited proteolysis of HtH1 withthe aid of elastase;

(b) SDS polyacrylamide gel electrophoresis (7.5% polyacrylamide) of theelastase-cleaved HtH1 subunit;

(c,d,g-j,l,n,p) Crossed immunoelectrophoresis of the elastase cleavageproducts of the HtH1 subunit;

(e) Crossed immunoelectrophoresis after limited proteolysis of HtH1 withthe aid of V8 protease;

(f) SDS polyacrylamide gel electrophoresis (7.5% polyacrylamide) of theV8 protease-cleaved HtH1 subunit;

(k,m,o) Crossed immunoelectrophoresis after limited proteolysis of HtH1with the aid of the three stated proteases.

FIG. 3 shows the separation of proteolytic cleavage products of thesubunit HtH1 with the aid of HPLC.

FIG. 4 shows the genomic sequence of the HtH1 gene.

FIG. 5 shows the primary structure deduced for the HtH1 protein.

FIG. 6 shows the genomic sequence of the HtH2 gene.

FIG. 7 shows the primary structure deduced for the HtH2 protein.

FIG. 8 shows the genomic sequence of the KLH1 gene.

FIG. 9 shows the primary structure deduced for the KLH1 protein.

FIG. 10 shows the genomic sequence of the KLH2 gene.

FIG. 11 shows the primary structure deduced for the KLH2 protein.

DETAILED DESCRIPTION OF THE INVENTION Examples

Material and Methods

1. Preparation of the Haemolymph and Isolation of Haemocyanin

Individuals of the European abalone Haliotis tuberculata from the FrenchAtlantic coast region were provided by S. M. E. L (Blainville sur Mer,France) and Biosyn (Fellbach, Germany). The animals were kept in a 300 1sea-water aquarium at 17° C. and fed with brown algae. For removal ofthe haemolymph, the abalones were placed on ice in a closed plastic bag.After one hour, large volumes of haemolymph had been secreted throughtheir skin. It emerged that the haemocyanin obtained by this process isidentical to the haemocyanin which could be collected by cutting ahollow in the foot of cooled-down sea snails using a scalpel blade. Theblood cells were separated from the haemolymph by centrifugation at 800g for 30 min at 4° C. The whole haemocyanin was then immediatelysedimented by preparative ultracentrifugation at 30,000 g for 4 hours at4° C. The supernatant was discarded and the blue haemocyanin pellet wassuspended overnight in “stabilization buffer” (0.05 M Tris, 5 mM CaCl₂,5 mM MgCl₂, 0.15 M NaCl, 1 mM PMSF, pH 7.4) and stored at 4° C.

Using the process described by Harris et al., 1995, supra, intact HtH1was obtained from the whole HtH by selective dissociation of HtH2 inammonium molybdate/polyethylene glycol (1%/0.2%) solution, pH 5.9 andsubsequent ultracentrifugation. The partly purified HtH1 pellet formedwas dissolved and purified to homogeneity by gel filtration on a BiogelA15m device. The last step resulted in small amounts of purified HtH2.Native HtH1 and HtH2 was dissociated quantitatively into the subunits bydialysis against “dissociation buffer” (0.13 M glycine/NaOH, pH 9.6) at4° C. overnight; the presence of EDTA was not necessary. 1 mM PMSF wasadded at each stage of the purification to inhibit proteolysis.

2. Electron Microscopy

Conventional “negative staining” was carried out by the individual dropmethod (Harris and Horne in Harris, J. R. (editors) Electron microscopyin biology, (1991), IRL Press Oxford, p. 203-228). Carbon carrier filmswere initially subjected to glow discharge for 20 seconds to render themhydrophilic and adsorptive for the protein. The protein samples areallowed to adsorb on to the carbon films for 60 seconds. The buffersalts are then removed by sequential washing with four successive 20 μldrops of water. Finally, the gratings are negatively stained with a 20μl drop of 5% aqueous ammonium molybdate containing 1% trehalose (pH7.0) and left to dry at room temperature. A Zeiss EM 900 transmissionelectron microscope is used for the electron microscopy analysis.

3. Polyacrylamide Gel Electrophoresis and Immunoelectrophoresis

SDS polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by themethod of Laemmli (Nature 227 (1970), 670-685). An alkaline systemaccording to Markl et al. (1979) J. Comp. Physiol. 133 B, 167-175 with a0.33 M Tris/borate, pH 9.6 as the gel buffer and 0.065 M Tris/borate, pH9.6 as the electrode buffer was used for the native PAGE. Crossed and“crossed-line” immunoelectrophoresis (1E) were carried out in accordancewith Weeke (Scand. J. Immunol. 2 (1973), Suppl. 1, 47-56) or Kroll(Scand. J. Immunol. 2, Suppl. 1 (1973), 79-81). Rabbit antibodiesagainst dissociated whole HtH and purified HtH1 were produced by CharlesRiver Deutschland (Kisslegg, Germany). The immunization process wascarried out in accordance with Markl and Winter (J. Comp. Physiol. 159B(1989), 139-151).

4. Limited Proteolysis and Isolation of the Fragments

The limited proteolysis was carried out at 37° C. in 0.13 Mglycine/NaOH, pH 9.6 by addition of one of the following enzymes (Sigma,Deisenhofen, Germany), which were dissolved in 0.1 M NH₄HCO₃, pH 8.0:Staphylococcus aureus V8 protease type XVII (8400), papain type II frompapaya milk (P-3125), bovine pancreas elastase type IV (E-0258),chymotrypsin and trypsin. The haemocyanin concentration was between 1and 10 mg/ml. The final concentration of the enzyme was 2%(weight/weight). The proteolysis was ended after 5 hours by freezing to−20° C. The HPLC process was carried out on a device from AppliedBiosystems (BAI, Bensheim, Germany) equipped with a model 1000S DiodeArray detector. The proteolytic fragments were introduced on to a smallMono-Q anion exchanger column (Pharmacia, Freiburg, Germany), which hadbeen equilibrated with 0.02 M Tris/HCl, pH 8.0, and were eluted with alinear sodium chloride gradient (0.0 M -0.5 M CaCl) in the same bufferat a flow rate of 1 ml/min. Alternatively, the proteolytic fragmentswere isolated by cutting out the bands from native PAGE gels (Markl etal., 1979) J. Comp. Physiol. 133 B, 167-175, after they had first beeninversely stained with the Roti-White system (Roth, Karlsruhe, Germany)in accordance with Fernandez-Patron et al. (1995) Anal. Biochem. 224,203-211. For subsequent cleavage with a second enzyme, the fragmentsisolated were first dialysed overnight against 0.13 M glycine/NaOH, pH9.6 to remove NaCl.

5. Amino Acid Sequence Analysis

The proteins obtained by the HPLC process were denatured inSDS-containing sample buffer and separated by SDS-PAGE (Laemmli, 1970,supra; 7.5% polyacrylamide). To prevent blocking of the NH₂ terminus,0.6% (weight/weight) thioglycollic acid was added to the cathode buffer(Walsh et al., Biochemistry 27 (1988), 6867-6876). The protein bandswere transferred by electro-transfer to ProBlot membranes (AppliedBiosystems, Germany) in a vertical blotting chamber (25 mM boratebuffer, pH 8.8, containing 2 mM EDTA; 10 min/100 mA, 15 min/200 mA, 12h/300 mA). Detection of the individual polypeptides on the membranes wascarried out with Ponceau S stain. The polypeptide bands of interest werecut out and sequenced in a 477A protein sequencing device from AppliedBiosystems. The amounts of polypeptides applied to the sequencing devicewere in the lower pmol range.

6. cDNA Cloning and Sequence Analysis

A lambda-cDNA expression library was established from poly(A⁺)—RNA fromHaliotis mantle tissue using the vector Lambda ZAP Express® inaccordance with the manufacturer's instructions (Stratagene, Heidelberg,Germany). The clones were isolated using HtH-specific rabbit antibodies.The nucleotide sequencing was carried out on both strands using the TaqDye deoxy Terminator® system. The sequences were arranged with thesoftware CLUSTAL W (1.7)® and TREEVIEW® (Thompson et al., Nucl. AcidsRes. 22 (1994), 4673-4680).

Example 1 Isolation of HtH and Separation of Two Different Types (HtH1and HtH2)

The haemolymph was obtained from adult abalones. The blood cells wereremoved by centrifugation and the haemocyanin was then sedimented byultracentrifugation. The blue haemocyanin pellet was dissolved again in“stabilization buffer” (pH 7.4) and examined by electron microscopy(FIG. 1 a). It comprised mainly typical di-decamers, accompanied by asmall content of decamers and tridecamers. Denaturing in 2% SDS in thepresence of reducing substances and subsequent SDS-PAGE separationresulted in a single band, which corresponded to the polypeptide with anapparent molecular weight of 370 kDa, which is only slightly below theapparent subunit weight of KLH (FIG. 1 b). Complete dissociation of theoligomers and of the di-decamers into the native polypeptides (subunits)was achieved by overnight dialysis of HtH against “dissociation buffer”(pH 9.6). The native PAGE method, which was used on these samples,showed a main and a secondary component (FIG. 1 c). Crossedimmunoelectrophoresis (crossed IE) using polyclonal rabbit antibodiesgenerated against purified whole HtH showed two components which areimmunologically different but show the classical reaction of beingpartly immunologically identical (FIG. 1 d). Their preparative isolation(FIG. 1 e-i) showed that they are subunits of two different HtH types,called HtH1 and HtH2, and the patterns of the native PAGE and crossed IEmethods could be assigned to each individually (FIG. 1 c, d).

The separation of HtH1 and HtH2 was carried out by the method ofselective dissociation according to Harris et al., 1995, supra. Inammonium molybdate/polyethylene glycol, HtH1 in the oligomer state(di-decamer) was completely stable, while HtH2 dissociated completelyinto the subunits (FIG. 1 e). This allowed quantitative sedimentation ofHtH1 in an ultracentrifuge, while the majority of the HtH2 remained inthe supernatant. Large amounts of HtH1 were purified to homogeneity fromthe redissolved pellet by gel filtration chromatography, which alsoresulted in small amounts of pure HtH2 (FIG. 1 f). The fractions wereinvestigated by native PAGE (FIG. 1 g) and crossed IE (FIG. 1 h, i). Theprocess of selective dissociation of HtH2 removed all the tri-decamerfrom the samples, which suggests that the latter are built up from HtH2,but not from HtH1 (FIG. 1 e). The selective dissociation behaviour ofHtH2 and also the ability to form aggregates which are larger than invivo di-decamers correspond to the properties of KLH2. Conversely, thestability of HtH1 under these conditions and its inability to assembleinto aggregates larger than di-decamers resemble the behaviour of KLH1.This feature of being related is demonstrated further by the reaction ofanti-KLH1 and anti-KLH2 antibodies against the two HtH types (FIG. 1j-m).

Example 2 Analysis of the Organization of the HtH1 Subunit

The eight functional units (FUs, often called “functional domains”)which form a mollusc haemocyanin subunit differ in primary structure andshow no immunological cross-reactivity, as emerged from crossed IE. Inthe case of the purified HtH1 subunit (FIG. 1 g, h), smallconcentrations of five different proteases (elastase, V8 protease,papain, trypsin and chymotrypsin) which had cleaved the peptide bondsbetween adjacent FUs of KLH1 and KLH2 were used (Gebauer et al., 1994,supra, Söhngen et al., 1997, supra). The cleavage products wereinvestigated by crossed IE and SDS-PAGE (FIG. 2). Elastase treatmentproduces eight individual FUs, deduced from the number of differentimmunoprecipitation peaks in the crossed IE (FIG. 2 a) and with theapparent molecular weight of approx. 50 kDa of the main portion of thecleavage products in SDS-PAGE (FIG. 2 b). A further precipitation peakwas recognized as FU dimer, which was formed by incomplete cleavage ofthe segment ab (FIG. 2 a). By an HPLC process with a Mono-Q column (FIG.3 a), two of the elastase cleavage products were obtained in asufficient purity to allow their clear assignment to two of the eightprecipitation peaks (FIG. 2 c, d) by “crossed-line IE”. The other fourproteases had different cleavage patterns, which comprised mixtures ofindividual FUs and larger fragments containing two, three or more FUs(e.g. FIG. 2 e, f). Many of them were concentrated to a sufficientamount by the HPLC process (FIGS. 3 b-e) to allow their identificationin their corresponding SDS-PAGE and crossed IE patterns. A number ofthese components were sequenced N-terminally by blot transfer of SDSgels on ProBlot® membranes (Table 1). The results were compared with theN-terminal sequences which had been obtained from the apparentlyorthologous protein in Megathura crenulata, KLH1 (Table I), the completeFU arrangement of which is available (Söhngen et al., 1997, supra; cf.FIG. 5 b). The result of the entire batch led to the determination ofthe complete FU arrangement within the HtH1 subunit (FIG. 2 a).

In particular, cleavage of the HtH1 subunit (1-abcdefgh) with V8protease resulted in four precipitation peaks in the crossed IE (FIG. 2e). The SDS-PAGE showed five different fragments (FIG. 2 f): 220 kDa (5FUs), 185 kDa (4 FUs), 100 kDa (2 FUs), 55 kDa (1 FU) and 46 kDa (1 FU).The 100 kDa fragment was isolated by the HPLC method (FIG. 3 b) andidentified by N-terminal sequencing as 1-ab, since the sequence wasidentical to that of the intact subunit (Table I). In the “crossed-line”IE process, 1-ab fused with three precipitation peaks of the elastasecleavage pattern. On the basis of the evaluation, they representfragments 1-ab, 1-a and 1-b (FIG. 2 g). However, it remained unclearwhich peak represents 1-a and which 1-b. In a second step, the 1-abpurified by HPLC was cleaved by elastase into its component FUs, fromwhich one could be eluted by the native PAGE gel strip method and wasassigned to the elastase pattern by the “crossed-line” IE method (FIG. 2h) and sequenced N-terminally. This component had the same N-terminalsequence as the whole subunit and was therefore identical to 1-a. Thesecond FU of the 100 kDa fragment is thus 1-b (FIG. 2 a; Table I).HPLC-purified 1-c and 1-h were also obtained (FIG. 3b), identified byN-terminal sequence similarities with the corresponding FUs in KLH1(Table I) and assigned by the “crossed-line” IE method to theircorresponding precipitation peaks in the elastase pattern (FIG. 2 i, j).1-a, 1-b, 1-c and 1-h were furthermore identified (FIG. 2 a). Usingpapain for subunit cleavage, five different peaks were obtained in thecrossed IE method (FIG. 2 k). A 100 kDa fragment (2 FUs) was purifiedfrom such a sample by the HPLC method (FIG. 3 c), and, according to the“crossed-line” IE method, contained the FU 1-h already identified andone of the four FUs still not identified and therefore must be 1-gh(FIG. 2 k, 3 c). In fact, this fragment had an N-terminal sequence whichshowed similarities with KLH1-g (Table I). For further confirmation, theHPLC-purified fragment 1-gh was cleaved into its constituent FUs withelastase, from which 1-g was purified and identified by N-terminalsequencing. It was assigned to its peak in the elastase cleavage patterby the “crossed-line” IE method (FIG. 21).

The 220 kDa fragment from the V8 protease cleavage (FIG. 2 e, f) waspurified by HPLC (FIG. 3 b) and in the “crossed-line” IE method fusedwith 1-h, 1-g and three peaks of the elastase cleavage pattern whichhave not yet been identified. The 185 kDa fragment was furthermoreobtained in a sufficient purity (FIG. 2 e, f; 3 b), and it was shownthat it comprised the same components with the exception of 1-h. Thissuggested that the 22 kDa and the 185 kDa fragment are 1-defgh and1-defg respectively. In fact, the N-terminal sequence was practicallyidentical and furthermore showed similarity with KLH1-d (Table I).Cleavage of the HtH1 subunit with trypsin resulted in a large number ofcomponents in the molecular weight range of one or two FUs (FIG. 2m).Several of the components were concentrated in HPLC fractions (FIG. 3d). A 100 kDa fragment proved to be particularly useful since it had thesame N-terminal sequence as the fragment 1-defg from the v8 proteasecleavage (Table I); the 100 kDa fragment should therefore be 1-de. Inthe “crossed-line” IE method, this component fused with two of the threeFU peaks of the elastase cleavage pattern not yet identified (FIG. 2 n),which should therefore be 1-d and 1-e, and thus left a singlepossibility for 1-f. The “crossed-line” IE method also showed that FU1-f was furthermore present in the 1-de fraction (FIG. 2n). Theidentification of 1-f was confirmed by cleavage of the subunit withchymotrypsin (FIG. 2 o) and a subsequent HPLC process (FIG. 3 e). Thiscleavage gave, inter alia, a 95 kDa fragment (2 FUs) which fused with1-g and a second peak (FIG. 2 p) in the “crossed-line” IE method andcould therefore be either 1-gh (which could be ruled out since 1-h hadalready been identified) or 1-fg (which seems appropriate on the basisof the further peak in question, which was identical to the remainingcandidate). In fact, this fragment showed a new N-terminal sequencewhich is similar to KLH1-f in a certain manner. The last problem was nowto assign the two remaining FU peaks to 1-d and 1-e. This was achievedusing HPLC-isolated FUs from samples in which the subunit had beencleaved with elastase. (FIGS. 2 c, d; 3 a). The more acidic component inthe crossed IE method was deduced as 1-d from its N-terminal sequence,which is identical to that of 1-defgh (FIG. 2 c, Table I), while themore basic component of the 1-d/1-g pair had a new N-terminal sequence(Table I) and therefore had to be 1-e (FIG. 2a). The structure of thefunctional units of subunit HtH1 was thus clarified.

Example 3

Comparison of the molecular weights and N-terminal sequences of thebiochemically isolated functional units (FUs) from HtH1 and KLH1. Thevarious FUs, each with an intact binuclear copper-binding site, wereliberated from their larger unit as globular segments by limitedproteolysis; cf. the section “Isolation and analysis of the units fromHtH1”. The KLH1 data were obtained from Söhngen et al., supra. Theassignment as an actual unit was done on the basis of the molecularweight and the immunological properties (cf. FIG. 2). The unusually lowmolecular weight of isolated HtH1-d could means that a large peptide wassplit off C-terminally.

TABLE 1 Functional unit Weight (kDa) N-terminal sequence HtH1-a 53DNVVRKDVSHLTDDEVQ KLH1-a 50 ENLVRKDVERL HtH1-b 48    ? KLH1-b 45    ?HtH1-c 46 FEDEKHSLRIRKNVDSLTPEENTNERLR KLH1-c 45 KVPRSRLIRKNVDRLTPSEHtH1-d 40 VEEVTGASHIRKNLNDLNTGEM KLH1-d 50 EVTSANRIRKNIENLS HtH1-e 49ILDHDHEEETLVRKNIIDLSP KLH1-e 50    ? HtH1-f 50KLNSRKHTPNRVRHELSSLSSRDIASLKA KLH1-f 45 HHLSXNKVRHDLSTL HtH1-g 45DHQSGSIAGSGVRKDVNTLTKAETDNLRE KLH1-g 45 SSMAGHFVRKDINTLTP HtH1-h 55DEHHDDRLADVLIRKEVDFLSLQEANAIKD KLH1-h 60 HEDHHEDILVRKNTHSL

Example 4 Cloning of Haemocyanin cDNA

1. For cloning the cDNA of haemocyanin, mRNA was isolated from themantle tissue of the particular mollusc. The first cDNA strand wasobtained by reverse transcription with Oligo(dT) as a primer. The secondstrand was obtained conventional synthesis with random primers. The cDNAobtained in this way was cloned in a lambda expression vector to form acDNA expression library. Using an anti-haemocyanin antibody, the librarywas searched under suitable conditions, positive clones being obtained.These positive clones were isolated, sequenced and characterized.

2. A cDNA probe was prepared from the N-terminal region of a positiveclone obtained, and the cDNA library was searched with this. Thepositive clones obtained were in turn isolated, sequenced andcharacterized.

3. To obtain sequences arranged still further to 5′, another expressionlibrary was established from cDNA, this being obtained with the aid of acombination of haemocyanin-specific and “random” primers. This cDNAlibrary was searched with cDNA probes which correspond to the“N-terminal” regions of the positive clones obtained under (2.). Thepositive clones obtained were isolated, sequenced and characterized.

Example 5 Cloning of Haemocyanin Genes

Genomic DNA was isolated by standard methods. The PCR reaction wascarried out with the aid of haemocyanin-specific primers in order toamplify the gene sections of the haemocyanins of interest. Theamplification products obtained were cloned in a suitable vector (forexample pgem T or pGem T easy (Promega, Mannheim) sequenced andcharacterized.

Example 6 Recombinant Expression of Haemocyanin

A PCR reaction was carried out with a cDNA clone which contains thecoding sequence for HtH-1d in order to amplify specifically the codingsequence of the domain 1d. Synthetically prepared oligonucleotides wereused as primers.

Primer 1 (upstream) comprises six nucleotides of the end of the domainHtH-1c, an SacI cleavage site and 12 nucleotides of the end of thedomain HtH-1d. Primer 2 (downstream) comprises six nucleotides of thestart of the domain HtH-1e, an SalI cleavage site and an HtH1-d-specificsequence.

PCR conditions:  2 min 95° C. 30 sec 95° C. 30 sec 55° C.  1 min 72° C.35 cycles 10 min 72° C.

The amplification product was cloned in the pGEM T easy PCR cloningvector (Promega) in XL-1 Blue (Stratagene). After isolation of therecombinant plasmid and restriction with SacI and SalI, the cDNA ofdomain 1d could be isolated. The expression vector pQE30 (Qiagen) wasalso restricted with the corresponding enzymes.

The ligation was then carried out between the HtH-1d-cDNA (restrictedwith SacI and SalI) and pQE (restricted with SacI and SalI). Directedcloning of the cDNA which codes for HtH-1d in an expression vector isthus possible. The expression of HtH1-d in pQE in XL-1 Blue is carriedout in accordance with the manufacturer's instructions. The expressionof further HtH1, HtH2 or KLH1 or KLH2 domains can be carried outanalogously.

1. An isolated nucleic acid molecule comprising a nucleic acid sequencethat codes for a Keyhole Limpit Hemocyanin 2 (KLH2) polypeptide andcomprises at least one intron sequence, wherein said nucleic acidsequence is selected from the group consisting of, (a) a DNA sequence orthe corresponding RNA sequence selected from the group consisting of:SEQ ID NO:20 (KLH2 domain b), SEQ ID NO:21 (KLH2 domain c), SEQ ID NO:22(partial KLH2 domain d), SEQ ID NO:23 (KLH2 domain g), SEQ ID NO:24(partial KLH2 domain h), SEQ ID NO:57 (KLH2 domain b′), SEQ ID NO:58(KLH2 domain c′), SEQ ID NO:59 (KLH2 domain d′), SEQ ID NO:60 (KLH2domain e), SEQ ID NO:61 (KLH2 domain f), SEQ ID NO:62 (KLH2 domain g′),SEQ ID NO:102 (KLH2 domain b″), SEQ ID NO:103 (KLH2 domain c″), SEQ IDNO:104 (KLH2 domain d″), SEQ ID NO:105 (KLH2 domain e″), SEQ ID NO:106(KLH2 domain f″), SEQ ID NO:107 (KLH2 domain g″); and, SEQ ID NO:108(partial KLH2 domain h″), (b) a nucleic acid sequence that hybridizesunder stringent hybridization conditions of 0.55×SSC; 1% blockingreagent; 0.1% sodium dodecyl sulfate (SDS) at about 68° C. overnight, toa counter-strand of a nucleic acid sequence according to (a); and (c) anucleic-acid sequence that has at least 90% sequence identity to one ofthe nucleic acid sequences described under (a).
 2. The isolated nucleicacid molecule according to claim 1, wherein the intron sequence isselected from: (i) a DNA sequencer the corresponding RNA sequenceselected from the group consisting of: SEQ ID NO:147 (KLH2 intron2B/2C), SEQ ID NO:148 (KLH2 intron 2C/2D), SEQ ID NO:149 (KLH2 intron2D/2E), SEQ ID NO:150 (KLH2 intron 2E/2F), SEQ ID NO:151 (KLH2 intron2F), SEQ ID NO:152 (KLH2 intron 2F-2/2G), SEQ ID NO:153 (KLH2 intron2G-1/2G-2), SEQ ID NO:154 (KLH2 intron 2G-2/2G-3), and SEQ ID NO:155(KLH2 intron 2G/2H); (ii) a nucleic acid sequence that hybridizes understringent conditions with the counter-strand of a nucleic acid sequenceaccording to (i); (iii) a nucleic acid sequence that has at least 90%sequence identity to one of the nucleic acid sequences described under(i); and (iv) combinations of several of the DNA sequences describedunder (i) to (iii).
 3. The isolated nucleic acid molecule according toclaim 1, wherein the nucleic acid molecule described under (b) has atleast 95% identity to a nucleic acid sequence described under (a). 4.The isolated nucleic acid molecule according to claim 2, wherein thenucleic acid molecule described under (iii) has at least 95% identity toone of the nucleic acid sequence described under (i).
 5. The isolatednucleic acid molecule according to claim 1 that is a deoxyribonucleicacid molecule.
 6. A process for the preparation of a KLH2 haemocyaninpolypeptide, wherein the nucleic acid molecule according to claim 1 isexpressed in a suitable host cell and the protein is isolated.
 7. Theprocess according to claim 6, wherein the KLH2 haemocyanin polypeptideprepared is modified naturally or chemically.
 8. The process accordingto claim 7, wherein the modification is cross-linking or covalentbonding to an antigen.
 9. The process according to claim 6, wherein theexpression is carried out in a host cell is a prokaryotic or eukaryoticcell suitable for expression of the construct.
 10. A compositioncomprising a nucleic acid molecule having a sequence according toclaim
 1. 11. A construct comprising a nucleic acid molecule sequencethat codes for a Keyhole Limpit Hemocyanin 2 (KLH2) polypeptide andcomprises at least one intron sequence wherein said nucleic acidsequence is selected from the group consisting of: (a) a DNA sequence orthe corresponding RNA sequence selected from the group consisting of:SEQ ID NO:20 (KLH2 domain b), SEQ ID NO:21 (KLH2 domain c), SEQ ID NO:22(partial KLH2 domain d), SEQ ID NO:23 (KLH2 domain g), SEQ ID NO:24(partial KLH2 domain h), SEQ ID NO:57 (KLH2 domain b′), SEQ ID NO:58(KLH2 domain c′), SEQ ID NO:59 (KLH2 domain d′), SEQ ID NO:60 (KLH2domain e), SEQ ID NO:61 (KLH2 domain f), SEQ ID NO:62 (KLH2 domain g′),SEQ ID NO:102 (KLH2 domain b″), SEQ ID NO:103 (KLH2 domain c″), SEQ IDNO:104 (KLH2 domain d″), SEQ ID NO:105 (KLH2 domain e″), SEQ ID NO:106(KLH2 domain f″), SEQ ID NO:107 (KLH2 domain g″), and SEQ ID NO:108(partial KLH2 domain h″); (b) a nucleic acid sequence that hybridizesunder stringent hybridization conditions of 0.55×SSC; 1% blockingreagent; 0.1% sodium dodecyl sulfate (SDS) at about 68° C. overnight, toa counter-strand of a nucleic acid sequence according to (a); and (c) anucleic-acid sequence that has at least 90% sequence identity to one ofthe nucleic acid sequences described under (a) (d) a combination of theDNA sequences described under (a) to (c).
 12. The construct according toclaim 11, further comprising a promoter suitable for expression control,the nucleic acid sequence that codes for a KLH2 polypeptide being underthe control of the promoter.
 13. The construct according to claim 11,further comprising a nucleic acid sequence that codes for an antigen andis coupled directly to the nucleic acid sequence that codes for a KLH2polypeptide.
 14. The construct according to claim 13, wherein theantigen is selected from the group consisting of: a tumour antigens, avirus antigens and an antigens of a bacterial or parasitic pathogen. 15.The construct according to claim 11, wherein the construct comprises atleast a part of a vector, the vector being selected from the groupconsisting of: a bacteriophag, an adenovirus, a vaccinia virus, abaculovirus, SV40 virus and a retrovirus.
 16. The construct according toclaim 11, wherein the construct further comprises a H is tag-codingnucleic acid sequence and the expression of the construct forms a fusionprotein with a H is tag.
 17. A isolated host cell containing a constructaccording to claim 11, wherein the host cell is a prokaryotic oreukaryotic cell suitable for expression of the construct.
 18. The hostcell according to claim 17, wherein the prokaryotic host cell isselected from E. coli and Bacillus subtilis.
 19. The host cell accordingto claim 17, wherein the eukaryotic host cell is selected from the groupconsisting of a yeast cell, a plant cell, an insect cell and a mammaliancell.
 20. The host cell according to claim 19, wherein the host cell isa CHO cell, a COS cell or a HeLa cell.
 21. A process for the preparationof a KLH2 haemocyanin polypeptide, wherein the construct-according toclaim 11 is expressed in a suitable host cell and the protein isisolated.